Oligonucleotides of known sequence are utilized in a wide variety of chemical and biological applications, including PCR (polymerase chain reaction) and cloning, as well as in the diagnosis and treatment of diseases (see, for example, Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993). It is often desirable to detect or isolate a specific, desired oligonucleotide from complex mixtures which may include oligonucleotide species having nucleobase sequences closely related to those of the desired oligonucleotide. This is especially important in biological samples, where the presence or absence of specific known nucleotide sequences can be indicative of presence or absence of an added oligonucleotide agent or, alternatively, a disease state. It is also very important in the manufacture of oligonucleotides for characterization of the purity of the product.
However, samples of interest often do not contain sufficient concentrations of oligonucleotides to permit detection by techniques such as ultraviolet (UV) spectroscopy or they contain other absorbing species that prohibit detection of the species of interest. Other analytic techniques may lack specificity for a particular nucleic acid sequence, or require excessive sample preparation or analysis times. Thus, there exists a need for a method, which does not have the aforementioned limitations, of detecting and isolation of oligonucleotides containing specific, known nucleotide sequences.
The use of electrophoretic techniques to separate oligonucleotide species is documented in the literature. One such technique is capillary electrophoresis (CE), which employs relatively long and thin capillary columns for the separation. See generally, Capillary Electrophoresis Theory and Practice, P. Grossman and L. Colburn, eds. Academic Press, New York (1992). The CE technique affords several advantages over conventional electrophoretic techniques such as polyacrylamide gel electrophoresis (PAGE). As CE is performed in very small diameter tubing (typically 50-100 .mu.m i.d.), electric fields 10 to 100 fold greater than those applicable in conventional electrophoretic systems can be applied because of reduced Joule heating. This affords very high speeds and superior resolution. Also, the CE technique lends itself to on-column detection by means such as ultraviolet spectroscopy, fluorescence spectroscopy, amperometric measurement, conductivity measurement or thermooptical detection. Additionally, CE can be performed with or without a gel medium in the capillary. When a gel medium such as polyacrylamide is employed, the technique is referred to a capillary gel electrophoresis (CGE).
There have been several reports of the use of CE in the detection of DNA species. For example, Luckey et al., Nucleic Acids Research 18 (15) 4417-4421 (1990) discloses a CE instrument developed for automated DNA sequencing in which products are detected via the fluorescence of an intercalating dye.
McCord et al., J. Chromatography 652 75-82 (1993) report the use of non-gel sieving buffers and fluorescent intercalating dyes in the CE analysis of PCR amplified DNA. Chen et al., Journal of Chromatography, 559, 295-305 (1991) describe the identification of DNA molecules by pre-column hybridization followed by capillary electrophoresis with on-line fluorescence detection.
Rose, Anal. Biochem. 65, 3545-3549 (1993) describes the use of CGE to separate peptide nucleic acid-oligonucleotide heteroduplexes from free single-strand oligonucleotide and single strand peptide nucleic acid (PNA). PNAs are capable of hybridization to complementary DNA or RNA sequences to form hybridized moieties which are more stable (i.e., which have higher binding affinities and higher melting temperatures) than corresponding "natural" duplexes. See Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993.
One disadvantage of the CE separation technique thus far has been the inherently irreproducible nature of the electrokinetic loading process. This results in the inability to quantitate the separated moieties. In the electrokinetic injection technique, the sample contents load onto the capillary both in response to the applied electric field, and by capillary action. Thus for most samples relatively small differences in the amounts of buffer salts present in the sample and an external standard can lead to dramatic differences in the amount of oligonucleotide loaded onto the column and the observed detector response. See Huang, X. et al., Anal. Chem. 60 375-377 (1988).
Thus, it would be of great advantage to have an analytical technique wherein the resolving power and superior resolution of CE could be applied in a quantitative fashion to the separation of oligonucleotide targets of similar length.